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United States Patent |
5,283,346
|
Barner
,   et al.
|
February 1, 1994
|
Dioxolanes
Abstract
Alcohols of the formula
##STR1##
wherein R.sup.1 and R.sup.2 are each independently methyl or ethyl or
together signify pentamethylene, formed by the enzymatic hydrolysis of a
corresponding (RS) alkanoic acid ester are intermediates for Vitamin E.
Inventors:
|
Barner; Richard (Witterswil, CH);
Hubscher; Josepf (Nunningen, CH);
Wirz; Beat (Reinach, CH)
|
Assignee:
|
Hoffmann-La Roche Inc. (Nutley, NJ)
|
Appl. No.:
|
990231 |
Filed:
|
December 14, 1992 |
Current U.S. Class: |
549/341; 549/342; 549/453; 549/454 |
Intern'l Class: |
C07D 317/72; C07D 317/18 |
Field of Search: |
549/341,342,453,454
|
References Cited
U.S. Patent Documents
4575558 | Mar., 1986 | Mai et al. | 549/453.
|
4822885 | Apr., 1989 | Banitt | 549/341.
|
4910220 | Mar., 1990 | Braga | 549/453.
|
4931575 | Jun., 1990 | Abushanab | 549/453.
|
Foreign Patent Documents |
0244912 | Nov., 1987 | EP.
| |
0388778 | Sep., 1990 | EP.
| |
0238232 | Aug., 1986 | DE.
| |
0257742 | Jun., 1988 | DE.
| |
Primary Examiner: Ivy; C. Warren
Assistant Examiner: Owens; A. A.
Attorney, Agent or Firm: Gould; George M., Epstein; William H.
Parent Case Text
This is a division of application Ser. No. 07/492,166 filed Mar. 13, 1992
now U.S. Pat. No. 5,232,852.
Claims
We claim:
1. The compound of the formula
##STR10##
where R1 and R2 are each independently methyl or ethyl or taken together
form pentamethylene; R3' is alkanoyl containing from 2 to 9 carbon atoms.
2. The compound of claim 1, wherein said compound is an RS racemate.
3. The compound of claim 2, wherein said compound is
{(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate.
4. The compound of claim 2, wherein said compound is
{(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate.
5. The compound of claim 1, wherein said compound is in the
(S)-enantiomeric form.
6. The compound of claim 5, wherein said compound is
{(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate.
7. The compound of claim 5, wherein said compound is
{(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate.
8. The compound {(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol.
9. The compound {(R)-2-methyl-1,4-dioxaspiro[4,5]-dec-2-yl}methanol.
10. The compound of formula
##STR11##
wherein R.sup.1 and R.sup.2 are each independently methyl or ethyl or
taken together form pentamethylene; and R.sup.3" is alkanesulfonyl or
arylsulfonyl.
11. The compound of claim 10, wherein said compound is in the
(R)-enantiomeric form.
12. The compound of claim 11, wherein said compound is
{(R)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl tosylate.
13. The compound of claim 11, wherein said compound is
{(R)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl tosylate.
14. The compound of claim 10, wherein said compound is in the
(S)-enantiomeric form.
15. The compound of claim 14, wherein said compound is
{(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl tosylate.
16. The compound of claim 14, wherein said compound is
{(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl tosylate.
Description
SUMMARY OF THE INVENTION
The present invention is directed to a process for producing chiral
dioxolane derivatives of the formula
##STR2##
wherein R.sup.1 and R.sup.2 are each independently methyl or ethyl or
taken together form pentamethylene, and to a class of new dioxolane
derivatives.
The compounds of formula Ib' and other members of the class of new
dioxolane derivatives are valuable intermediates in the manufacture of
vitamin E, especially of its optically active form d-.alpha.-tocopherol.
DETAILED DESCRIPTION
In accordance with this invention the compound of formula Ib' is produced
by hydrolyzing a racemic alkanoic acid ester of the formula
##STR3##
wherein R.sup.1 and R.sup.2 are as above, and R.sup.3' is alkanoyl
containing from 2 to 9 carbon atoms; through enzymatic hydrolysis using an
enzyme of the sub-class carboxyl esterases (EC 3.1.1.1), triacylglycerol
lipases (EC 3.1.1.3), cholesterol esterases (EC 3.1.1.13) or
diacylglycerol lipases (EC 3.1.1.34). The so-produced alcohol of formula
Ib' or the (S)-alkanoic acid ester of Ia, which is not reacted during the
enzymatic hydrolysis in accordance with the invention, can be converted
into d-.alpha.-tocopherol in a manner which will be explained in more
detail hereinafter.
The hydrolysis is carried out in accordance with the invention under the
influence of an enzyme of the above-mentioned sub-classes, preferably a
lipase of microbial origin from Pseudomonas fluorescens, Mucor miehei,
Rhizopus delemar, Rhizopus arrhizus, Humicola lanuginosa, Rhizopus
javanicus or Mucor javanicus. Examples of suitable enzyme preparations are
Lipase P-30 (P. fluorescens; Amano Pharmaceutical Co. Ltd., Nagoya,
Japan), Lipase SAM (P. fluorescens; Amano; available under catalog No.
62312 from Fluka AG, Buchs, Switzerland), Lipase PM and LMM (M. miehei;
Palatase M 1000L or Lipozyme IM 20; Novo Industri A/S, Bagsvaerd,
Denmark), Lipase LRD and D (R. delemar; Seikagaku Kogyo Co. Ltd., Tokyo,
Japan or Amano), Lipase LRA (R. arrhizus; Sigma Chemical Co., St. Louis,
USA), Lipase CE (H. lanuginosa; Amano), Lipase FAR (R. javanicus; Amano)
and Lipase MAP (M. javanicus; Amano).
The enzyme can be used not only in partially purified or completely pure
form, but also in immobilized form.
The process in accordance with the invention is conveniently carried out in
an organic/aqueous emulsion in which the organic phase can consist solely
of the starting material of formula Ia, but which can optionally also
contain a water-immiscible, non-polar or polar solvent such as, for
example, n-hexane, isooctane or diethyl ether. One of the usual inorganic
or organic buffers, e.g. sodium phosphate or sodium citrate, can be
present in the aqueous phase, conveniently in a molar concentration of 1
mM to 1.0M, preferably 3 mM to 0.1M.
Moreover, the process in accordance with the invention can be carried out
in the presence of a monohydric or polyhydric alcohol, e.g. ethanol or
glycerol, conveniently at a molar concentration of 50 mM to 4M, preferably
50 mM to 0.5M; of calcium or magnesium ions; a commercial emulsifier, e.g.
polyvinyl alcohol or Triton.RTM. X-100; and/or a "salting-in" salt, e.g.
lithium rhodanide or guanidinium chloride, conveniently at a molar
concentration of 50 mM to 2M, preferably 50 mM to 0.5M.
The concentration of the substrate (alkanoic ester of formula Ia) in the
total reaction medium is conveniently 0.5% to 50%, preferably 1% to 15%,
with the percentage concentrations referring to weight/volume (w/v). The
weight ratio enzyme:substrate conveniently amounts to 0.001-0.05:1.
The hydrolysis in accordance with the invention is conveniently effected at
temperatures between 0.degree. and 45.degree. C., preferably between
10.degree. and 35.degree. C. The hydrolysis is conveniently carried out at
a pH value of 6 to 9, preferably 7 to 8.
Starting from racemic (RS)-alkanoic ester Ia there is obtained initially as
the reaction product predominantly the (S)-alcohol of formula Ib', i.e.
the process in accordance with the invention is an asymmetric hydrolysis
in that the (R)-ester of the racemic mixture Ia can hydrolyze to the
(S)-alcohol Ib' under the influence of the enzyme much more rapidly than
the (S)-ester can hydrolyze to the (R)-alcohol of the formula
##STR4##
Thereby, at up to a 50 percent reaction conversion the by far predominant
part of the (S)-ester, i.e. of the formula
##STR5##
of the racemic mixture Ia remains behind. It has been found that at up to
a 50 percent reaction conversion the ratio alcohol Ib'
[(S)-alcohol]:alcohol Ib" [(R)-alcohol], i.e. the so-called ee value
(enantiomeric excess or enantiomeric purity), remains extremely high,
namely at at least 90%, in most cases at about 98%. Herein lies the
advantage of the process in accordance with the invention. A further
material advantage of the process in accordance with the invention is that
with the formation of the product Ia' no mere byproduct arises, but rather
a product (of formula Ia') which can also be converted into
d-.alpha.-tocopherol.
After the 50 percent reaction conversion has been achieved, i.e. at the
point in time when the conversion of the (R)-ester present in the racemate
Ia to the (S)-alcohol Ib' has been completed (which can be established by
conventional analytical methods, e.g. titration), the enzymatic hydrolysis
reaction can be stopped by conventional means, for example by the addition
of an organic solvent, e.g. methylene chloride or methyl tert.butyl ether.
The (S)-alcohol and the unreacted (S)-ester are then isolated from the
reaction mixture, e.g. by extraction with an organic solvent such as, for
example, chloroform or methyl tert.butyl ether and subsequent evaporation
of the solvent, and the two components are separated from one another,
e.g. by fractional distillation or column chromatography.
As mentioned above, the compounds of formulae Ia' and Ib' are valuable
intermediates in the manufacture of optically active d-.alpha.-tocopherol.
The respective synthetic approaches which can be carried out using these
intermediates are illustrated in the following Reaction Scheme:
##STR6##
In the above Reaction Scheme R.sup.3' signifies C.sub.2-9 -alkanoyl, i.e.
acetyl to nonanoyl, with n-butyryl being preferred; and R.sup.3" signifies
alkanesulfonyl or arylsulfonyl, preferably methanesulfonyl (mesyl) or
p-tolylsulfonyl (tosyl). The compounds of formulae II and Ia occur as
racemates [(RS)-forms], while the compounds of formulae Ib", Ic' and III
are present in the (R)-enantiomeric form and the compounds of formulae
Ia', Ib', Ic" and IV are present in the (S)-enantiomeric form. In these
formulae it is to be understood by the symbol or that the substituent
CH.sub.3, CH.sub.2 OR.sup.3', CH.sub.2 OH or CH.sub.2 OR.sup.3" lies below
and, respectively, above the plane of the molecule.
The (RS)-alcohols of the formula II, which are used as starting materials,
are to some extent known (see e.g. J. Org. Chem. 1983, 45, 3592-3594, and
J. Chem. Soc., Chem. Commun., 1987, 538-539, in which there are described
those (RS)-compounds II in which not only R.sup.1 but also R.sup.2
signifies methyl). The remaining, i.e. novel, (RS)-alcohols of formula II,
i.e. those compounds of formula II in which R.sup.1 and R.sup.2 have the
significances given above although do not both signify methyl, can be
produced according to methods known per se.
Most of the intermediates of formulae Ia, Ia', Ib', Ib", Ic' and Ic" are
novel and form a further object of the present invention. These novel
compounds are embraced by formula I hereinafter:
##STR7##
wherein R.sup.1 and R.sup.2 each independently signify methyl or ethyl or
together signify pentamethylene and R.sup.3 signifies hydrogen, C.sub.2-9
-alkanoyl, alkanesulfonyl or arylsulfonyl, with the provisos that R.sup.3
is different from hydrogen where R.sup.1 and R.sup.2 both stand for
methyl, and that the compound I is chiral where R.sup.1 and R.sup.2
together stand for pentamethylene and R.sup.3 stands for hydrogen.
Under the compounds of formula I there are accordingly to be understood not
only the chiral compounds [(R)- or (S)-enantiomeric form] but also
mixtures of the two forms, i.e. the racemates, and
{2-methyl-1,4-dioxaspiro[4,5]-dec-2-yl}methanol (formula I in which
R.sup.1 and R.sup.2 together signify pentamethylene and R.sup.3 signifies
hydrogen) as the racemate is excluded.
From the definition of the compounds of formula I and the above Reaction
Scheme it will be evident that the novel compounds of formula I in
accordance with the invention consist of the following sub-classes:
The compounds of the formula 1a in which R.sup.1 and R.sup.2 have the
significances given above and R.sup.3' signifies C.sub.2-9 -alkanoyl
[among these compounds there are to be found especially the racemates
[(RS)-form], which are the products of the reaction of the racemic
compounds of formula II with the respective acid anhydride
(R.sup.3').sub.2 O, as well as the compounds of formula Ia', i.e. the
(S)-enantiomers];
The compounds of the formula
##STR8##
wherein R.sup.1 and R.sup.2 each independently signify methyl or ethyl or
together signify pentamethylene, with the provisos that R.sup.1 and
R.sup.2 do not both stand for methyl, and that the compound Ib is chiral
where R.sup.1 and R.sup.2 together stand for pentamethylene, i.e. is
present either in the (R)-enantiomeric form or in the (S)-enantiomeric
form [among these compounds there are to be found especially compounds of
formula Ib', i.e. the (S)-enantiomers, as well as compounds of formula
Ib", i.e. the (R)-enantiomers]; as well as the compounds of the formula
##STR9##
wherein R.sup.1 and R.sup.2 each independently signify methyl or ethyl or
together signify pentamethylene and R.sup.3" signifies alkanesulfonyl or
arylsulfonyl, among which there are to be found especially the compounds
of formula Ic', i.e. the (R)-enantiomers, as well as the compounds of
formula Ic", i.e. the (R)-enantiomers.
In accordance with the definition formulae Ib' and Ib" embrace not only
novel compounds, but also the known compounds of formulae Ib' and Ib" in
which R.sup.1 and R.sup.2 both stand for methyl (see J. Org. Chem. 1983,
48, 3592-3594 and J. Chem. Soc. Chem. Commun., 1987, 538-539).
Under the term "C.sub.2-9 -alkanoyl" there are to be understood not only
straight-chain but also branched alkanoyl groups. This also applies to
"alkanesulfonyl" (R.sup.3"), with the alkane part being especially
C.sub.1-4 -alkyl. The arylsulfonyl group (R.sup.3") is especially
phenylsulfonyl, tolylsulfonyl or naphthylsulfonyl. As mentioned above, the
preferred C.sub.2-9 -alkanoyl, alkanesulfonyl or arylsulfonyl group is,
respectively, n-butyryl, methanesulfonyl (mesyl) or p-toluenesulfonyl
(tosyl).
Especially preferred individual compounds in accordance with the invention
are:
Formula Ia or Ia':
{(R,S)-2-Methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate,
{(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate,
{(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate and
{(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate.
Formula Ib' or Ib":
{(S)-2-Methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol and
{(R)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol.
Formula Ic' or Ic":
{(R)-2-Methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl tosylate,
{(R)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl tosylate,
{(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl tosylate and
{(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl tosylate.
Having regard to the carbon number of the basic structure the compounds I
can be described as optically active C.sub.4 -building blocks.
The two products of the hydrolysis in accordance with the invention are the
(S)-alcohol Ib' and the (S)-ester Ia', with the latter product being a
(unreacted) component of the racemic starting material Ia. The known
optically active diols of formulae III and IV can be produced from the two
isolated products (see the Reaction Scheme). The one synthetic route is a
conventional esterification of the (S)-alcohol Ib' to its alkanesulfonate
or arylsulfonate of formula Ic', whereby the invension S.fwdarw.R takes
place, followed by a cleavage of the protecting group R.sup.1 R.sup.2 C<
which again is carried out under reaction conditions which are familiar to
a person skilled in the art. In the case of the second synthetic route,
the (S)-ester Ia' must firstly be hydrolyzed to the (R)-alcohol Ib", which
can be effected under reaction conditions which are familiar to the person
skilled in the art. Thereafter, the (R)-alcohol can be converted into the
diol of formula IV by esterification and subsequent cleavage of the
protecting group R.sup.1 R.sup.2 C<, namely analogously to reaction steps
Ib'.fwdarw.Ic' and Ic'.fwdarw.III. Thus, the esterification
(Ib'.fwdarw.Ic' or Ib".fwdarw.Ic") is conveniently effected in an inert
organic solvent. preferably a halogenated aliphatic hydrocarbon,
especially methylene chloride, at temperatures between 0.degree. C. and
room temperature. The alkanesulfonating or arylsulfonylating agent is
suitably the corresponding sulfonic acid chloride such as, for example,
methanesulfonyl chloride (mesyl chloride) or p-toluenesulfonyl chloride
(tosyl chloride). Moreover, the presence of a base such as a tertiary
amine, preferably triethylamine, is advantageous. The subsequent cleavage
of the protecting group (Ic'.fwdarw.III or Ic".fwdarw.IV) is also
conveniently carried out in an inert organic solvent, preferably in a
lower alcohol such as a methanol. Moreover, the reaction is suitably
effected in the presence of a catalytic amount of a sulfonic acid such as
p-toluenesulfonic acid. A preferred method comprises subjecting the
protected compound (Ic' or Ic") to a trans-ketalization. For example, a
1,4-dioxaspiro[4,5]decane derivative of formula Ic' or Ic" (R.sup.1 and
R.sup.2 together signify pentamethylene) is repeatedly evaporated from
methanol in the presence of p-toluene-sulfonic acid, whereby the diol
product III or IV finally results.
The diols of formulae III and IV are known compounds (see, for example,
European Patent Publications Nos. 257 503 and 269 009) and can be
converted into d-.alpha.-tocopherol according to methods known per se
(see, for example, the two above-mentioned European Patent Publications as
well as European Patent Publication No. 129 252). The conversion of the
S-diol (formula IV) into d-.alpha.-tocopherol can be effected by coupling
this diol with the aromatic ring, subjecting the product to a ring closure
and subsequently attaching the C.sub.15 -side chain or by carrying out the
latter two reaction steps in reverse sequence. On the other hand, when
using the R-diol (formula III), this is firstly coupled with the C.sub.15
-side chain in order to obtain the "C.sub.19 -building block". This is
then linked benzylically with the aromatic ring and the ring closure is
carried out. The details for the various reaction steps are to be found in
the above-mentioned Patent Publications.
The conversion in accordance with the invention into d-.alpha.-tocopherol
is carried out either by esterifying the alcohol of formula Ib', which is
manufactured by the hydrolysis in accordance with the invention, to the
corresponding (R)-alkanesulfonate or (R)-arylsulfonate of formula Ic',
cleaving off the protecting group R.sup.1 R.sup.2 C< and converting the
resulting (R)-diol of formula III into d-.alpha.-tocopherol in a manner
known per se or by hydrolyzing the (S)-alkanoic acid ester of formula Ia',
which remains in the starting material during the enzymatic hydrolysis in
accordance with the invention, to the corresponding (R)-alcohol of formula
Ib", esterifying this (R)-alcohol to the corresponding (S)-alkanesulfonate
or (S)-arylsulfonate of formula Ic", cleaving off the protecting group
R.sup.1 R.sup.2 C< and converting the resulting (S)-diol of formula IV
into d-.alpha.-tocopherol in a manner known per se.
The following Examples illustrate the invention.
EXAMPLE 1
Production of {(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate
5.62 g (26.7 mmol) of (R,S)-2-methyl-1,4-dioxaspiro[4,5]decane-2-methanol
are dissolved in 20 ml of methylene chloride, 4.57 ml (3.32 g, 32.8 mmol)
of triethylamine, 4.90 ml (4.75 g, 30.0 mmol) of butyric anhydride and a
catalytic amount of 4-dimethylaminopyridine are added to the solution
while stirring and the reaction mixture is subsequently stirred at room
temperature for a further 5 hours. The mixture is then washed with water,
the organic phase is concentrated under reduced pressure and the residue
is purified by chromatography over silica gel 60 (0.040-0.063 mm, 300 g)
using n-hexane/ethyl acetate (4:1) as the eluent. In this manner there are
obtained 6.40 g (25.0 mmol, 93.5% of the theoretical yield) of the
butyrate as a colourless oil which by gas chromatography is found to be
more than 99% pure and which contains no traces of educt alcohol.
EXAMPLE 2
Production of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate
17.3 g (118.3 mmol) of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol
are dissolved in 70 ml of methylene chloride. 23.0 ml (16.7 g, 165.0 mmol)
of triethylamine, 19.4 ml (18.8 g, 118.7 mmol) of butyric anhydride and a
catalytic amount of 4-dimethylaminopyridine are added to the solution
while stirring and the reaction mixture is subsequently stirred at room
temperature for a further 5 hours. The mixture is then washed with water,
the organic phase is concentrated under reduced pressure and the residue
is purified by chromatography over silica gel 60 (0.040-0.063 mm) using
n-hexane/ethyl acetate (3:1) as the eluent. In this manner there are
obtained 23.5 g (108.6 mmol, 91.8% of the theoretical yield) of the
butyrate as a colourless oil which by gas chromatography is found to be
more than 99% pure and which contains no traces of educt alcohol.
EXAMPLE 3
Manufacture of {(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol
(asymmetric hydrolysis of
{(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate)
100 mg (390 .mu.mol) of {(RS)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl
butyrate are emulsified in 25 ml of 0.1M sodium chloride solution and 1 ml
of 0.1M sodium phosphate buffer at pH 7. After adjusting the pH value to
7.5 (or 7.0) with 0.1N sodium hydroxide solution the reaction is initiated
by adding a catalytic amount of the respective enzyme. The pH value is
held constant at 7.5 or 7.0 by stirring in 0.1N sodium hydroxide solution
up to about 50 percent reaction conversion [after the addition of about
195 .mu.mol of NaOH (50% ester equivalents, 1.95 ml)]. Thereafter, the
reaction is interrupted by the addition of 25 ml of methylene chloride.
The reaction mixture is extracted with 25 ml of methylene chloride, the
phase separation being accelerated by centrifugation for a short time, the
organic phase is dried over anhydrous magnesium sulfate, the solvent is
evaporated off and the residue is subjected to capillary gas
chromatography for the direct determination of the enantiomeric purity of
the alcohol product (by means of chiral phase: permethylated
.beta.-cyclodextrin).
The enzymes used, the percentage conversion, the corresponding reaction
time as well as the percentage enantiomeric purity (ee) of the
thus-produced {(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol are given
in Table 1 hereinafter.
TABLE 1
______________________________________
Percentage
Enzyme (amount
conversion/ Percentage ee of
used)* reaction time the alcohol product
______________________________________
Lipase P-30 (200 U)
48.6/35 min. 99
Lipase SAM (85 U)
48.1/168 min.**
99
Lipase PM about 50/40 min.
98
[Palatase M 1000 L]
(100 .mu.l)
Lipase LMM about 49 91
[Lipozym IM 20]
Lipase LRD (525 U)
48.8/52 min. 98
Lipase D-20 (14 mg)
49.2/38 min. 97
Lipase LRA (1481 U)
49.5/15 min. 97
Lipase CE-5 (23 mg)
49.0/10 min. 98
Lipase F-AP 15
47.6/176 min.**
95
(1008 U)
Lipase M-AP 10
48.7/168 min. 96
(80 U)
______________________________________
*Amount in mg, .mu.l or U (amount of units used according to details
declared by the supplier)
**Reaction carried out at PH 7.0 (otherwise 7.5)
EXAMPLE 4
Manufacture of {(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol
(asymmetric hydrolysis of
{(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate, alternative
method)
The procedure of Example 3 is repeated with the differences that 1.0 g
(3.91 mmol) or 3.0 g (11.73 mmol) of the butyrate are used instead of 100
mg (390 .mu.mol), and 1.0N sodium hydroxide solution is used instead of
0.1N sodium hydroxide solution as the titrating agent.
The respective details are set forth in Table 2 hereinafter:
TABLE 2
______________________________________
Amount of Percentage Percentage ee
Enzyme (amount
butyrate conversion/
of the alcohol
used)*** used reaction time
product
______________________________________
Lipase P-30 1.0 g 47.3/150 min.
99.2
(600 U)
Lipase P-30 3.0 g 48.9/200 min.
99.1
(1800 U)
Lipase PM 1.0 g 45.1/154 min.
97.3
[Palatase M 1000 L]
(300 .mu.l)
Lipase PM 3.0 g 47.2/316 min.
96.0
[Palatase M 1000 L]
(1.0 ml)
Lipase LRD 1.0 g 49.8/184 min.
97.7
(1575 U)
Lipase LRD 3.0 g 48.6/370 min.
95.9
(5250 U)
______________________________________
***Amount in ml, .mu.l or U (amount of units used according to details
declared by the supplier)
EXAMPLE 5
Manufacture of {(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol
(asymmetric hydrolysis of
{(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate)
The procedure of Example 3 is repeated using Lipase P-30 (200 U) at pH 7.5,
but with the difference that
0.1M lithium rhodanide is used in place of 0.1M sodium chloride (variant
A),
0.1M glycerol is used in place of 0.1M sodium chloride (variant B),
0.1M calcium chloride is used in place of 0.1M sodium chloride (variant C),
0.1M magnesium chloride is used in place of 0.1M sodium chloride (variant
D) or
the aqueous phase additionally contains 26 .mu.l of Triton X-100 (variant
E).
The respective details are set forth in Table 3 hereinafter:
______________________________________
Percentage ee
Percentage of the alcohol
Variant conversion Reaction time
product
______________________________________
A 49.5 41 min. 99
B 49.6 48 min. 99
C 49.5 36 min. 99
D 49.6 29 min. 99
E 49.5 59 min. 99
______________________________________
EXAMPLE 6
Manufacture of {(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol (asymmetric
hydrolysis of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate)
100 mg (462 .mu.mol) of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl
butyrate are hydrolyzed to the corresponding methanol derivative
analogously to the procedure described in Example 3. For the determination
of the enantiomeric purity of the alcohol product by capillary gas
chromatography (permethylated .beta.-cyclodextrin) it is previously
converted into the corresponding benzoate using benzoyl chloride.
The enzymes used, the percentage conversion, the corresponding reaction
time as well as the percentage enantiomeric purity (ee) of the
thus-manufactured {(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol are
given in Table 4 hereinafter:
TABLE 4
______________________________________
Percentage
Enzyme (amount
conversion/ Percentage ee of the
used)* reaction time
alcohol product
______________________________________
Lipase P-30 49.5/26 min.
99.6
(2OO U)
Lipase SAM (85 U)
49.5/81 min.
99.6
Lipase PM 49.6/26 min.
99.4
[Palatase M 1000 L]
(100 .mu.l)
Lipase LRD (525 U)
49.5/28 min.
98.2
Lipase D-20 49.5/24 min.
97.0
(14 mg)
Lipase F-AP 15
49.5/16 min.
97.1
(1008 U)
Lipase M-AP 10
49.5/60 min.
96.6
(80 U)
______________________________________
*Amount in mg, .mu.l or U (amount of units used according to details
declared by the supplier)
EXAMPLE 7
Manufacture of {(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol
(asymmetric hydrolysis of
{(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate)
5.00 g (19.5 mmol) of {(R,S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl
butyrate are emulsified in 100 ml of 0.1M sodium chloride solution and 4
ml of 0.1M sodium phosphate solution at pH 7 (concentration of butyrate
about 4.6% w/v). The reaction is initiated by the addition of 74.0 mg of
Lipase P-30. The pH value is held constant at 7.0 by stirring in 1.0N
sodium hydroxide solution up to 45.6 percent reaction conversion (reaction
time about 2.25 hours, addition of 8.90 ml of sodium hydroxide solution).
Thereafter, the reaction is interrupted by the addition of 50 ml of
methylene chloride. The reaction mixture is extracted twice with 50 ml of
methylene chloride each time, the phase separation being accelerated by
centrifugation for a short time, the combined organic phases are dried
over anhydrous magnesium sulfate and the solvent is evaporated off at 16
mmHg/room temperature. This gives 4.44 g of a colorless oil which is
purified by chromatography over silica gel 60 (0.040-0.063 mm, 99 g) using
500 ml of methylene chloride followed by 500 ml of methylene
chloride/diethyl ether (1:1) as the eluent. In this manner there are
obtained 1.80 g (8.56 mmol, 88% of the theoretical yield) of
{(S)-2-methyl-1,4-dioxaspiro[4,5]-dec-2-yl}methanol, >99% pure by gas
chromatography, >99% enantiomeric purity (ee), [.alpha.].sub.365
=-20.1.degree. (1% in CHCl.sub.3) and +20.9.degree. (1% in C.sub.2 H.sub.5
OH).
[The likewise isolated ester fraction
({(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl butyrate) can be
concentrated and hydrolyzed in alkaline solution to
{(R)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl)methanol].
EXAMPLE 8
Manufacture of {(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol (asymmetric
hydrolysis of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate,
alternative method)
The procedure of Example 6 is repeated, but with the differences that 1.0 g
(4.6 mmol) or 3.0 g (13.9 mmol) or 5.0 g (23.1 mmol) of the butyrate are
used instead of 100 mg (462 .mu.mol), and 1.0N sodium hydroxide solution
is used as the titrating agent.
The respective details are set forth in Table 5 hereinafter:
TABLE 5
______________________________________
Amount of Percentage Percentage ee
Enzyme butyrate conversion/
of the alcohol
(amount used)***
used reaction time
product
______________________________________
Lipase P-30 1.0 g 49.5/28 min.
99.4
(600 U)
Lipase P-30 3.0 g 49.3/60 min.
99.0
(1800 U)
Lipase P-30 5.0 g 48.9/120 min.
98.8
(1800 U)
Lipase PM 1.0 g 49.5/90 min.
97.3
[Palatase M 1000 L]
(300 .mu.l)
Lipase LRD 1.0 g 49.5/132 min.
94.4
(1575 U)
______________________________________
***Amount in .mu.l or U (amount of units used according to details
declared by the supplier)
EXAMPLE 9
Manufacture of {(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol (asymmetric
hydrolysis of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate
5.01 g (23.2 mmol) of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl
butyrate are emulsified in 25 ml of 0.1M sodium chloride solution and 1 ml
of 0.1M sodium phosphate buffer at pH 7.5 (concentration of butyrate 16.1%
w/v). The reaction is initiated by adding 50 mg of Lipase P-30. The pH
value is held constant at 7.5 by stirring in 1.0N sodium hydroxide
solution up to a 46.6 percent reaction conversion (reaction time 1 hour,
addition of 10.8 ml of sodium hydroxide solution). Thereafter, the
reaction is interrupted by the addition of 50 ml of methylene chloride.
The reaction mixture is extracted twice with 50 ml of methylene chloride
each time, the phase separation being accelerated by centrifugation for a
short time, the combined organic phases are dried over anhydrous magnesium
sulfate and the solvent is evaporated off at 12 mbar/30.degree. C. This
gives 4.07 g of an oil which is purified by chromatography over silica gel
60 (0.040-0.063 mm, 80 g) using n-hexane/ethyl acetate (2:1) as the
eluent. In this manner there are obtained 1.33 g (9.11 mmol, 78.6% of the
theoretical yield) of {(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol,
>99% pure according to gas chromatography, >99% enantiomeric purity (ee),
[.alpha.].sub.365 =-24.2.degree. (1% in CHCl.sub.3) and, respectively,
[.alpha.].sub.365 =+26.0.degree. (0.5% in ethanol).
EXAMPLE 10
Production of {(R)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl tosylate
A solution of 1.67 g (7.94 mmol) of
{(S)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methanol and 3 ml of
triethylamine in 10 ml of methylene chloride is treated portionwise with
1.63 g (8.54 mmol) of tosyl chloride and the reaction mixture is left to
stand for about 16 hours. The resulting brown mass is taken up in 100 ml
of methylene chloride and the solution is washed twice with 100 ml of
water each time. Subsequent evaporation of the organic phase gives 3.1 g
of a brownish oil which is purified by chromatography over silica gel 60
(0.040-0.063 mm, 70 g) using methylene chloride as the eluent. There are
obtained 2.33 g (6.84 mmol, 86% of the theoretical yield) of
{(R)-2-methyl-1,4-dioxaspiro[4,5]-dec-2-yl}methyl tosylate, 99% pure by
gas chromatography, [.alpha.].sub.365 =-29.0.degree. (1% in CHCl.sub.3).
EXAMPLE 11
Production of {(R)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl tosylate
A solution of 1.15 g (7.86 mmol) of
{(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol and 3 ml of triethylamine
in 25 ml of methylene chloride is treated portionwise with 1.90 g (9.96
mmol) of tosyl chloride and the reaction mixture is left to stand for
about 16 hours. The reaction mixture is washed twice with 100 ml of water
each time. Subsequent evaporation of the organic phase gives 3.04 g of a
brownish oil which is purified by chromatography over silica gel 60
(0.040-0.063 mm, 80 g) using n-hexane/ethyl acetate (3:1) as the eluent;
the first tosylate fractions obtained are again purified by chromatography
using n-hexane/ethyl acetate (2:1) as the eluent. A total of 1.78 g (5.92
mmol, 75% of the theoretical yield) of
{(R)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl tosylate is obtained, 99%
pure by gas chromatography, [.alpha.].sub.365 =-37.1.degree. (1% in
CHCl.sub.3).
EXAMPLE 12
Production of (R)-2,3-dihydroxy-2-methylpropyl tosylate
2.22 g (6.52 mmol) of {(R)-2-methyl-1,4-dioxaspiro[4,5]dec-2-yl}methyl
tosylate are repeatedly evaporated from methanol in the presence of a
catalytic amount of p-toluenesulfonic acid and the residue is finally
purified by chromatography over silica gel 60 (0.040-0.063 mm, 55 g) using
diethyl ether/methylene chloride (1:2) as the eluent. There are obtained
829 mg (3.18 mmol, 49% of the theoretical yield) of
(R)-2,3-dihydroxy-2-methylpropyl tosylate, 98% pure by gas chromatography,
[.alpha.].sub.365 =-19.9.degree. (1% in CHCl.sub.3).
EXAMPLE 13
Production of (R)-2,3-dihydroxy-2-methylpropyl tosylate
1.42 g (4.74 mmol) of {(R)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl
tosylate are evaporated twice from 100 ml of methanol in the presence of a
catalytic amount of p-toluenesulfonic acid at 16 mbar/40.degree. C. and
the residue is finally purified by chromatography over silica gel 60
(0.040-0.063 mm, 20 g) using methylene chloride/diethyl ether (1:1) as the
eluent. There are obtained 907 mg (3.49 mmol, 74% of the theoretical
yield) of (R)-2,3-dihydroxy-2-methylpropyl tosylate, 99% pure by gas
chromatography, [.alpha.].sub.365 =-19.0.degree. (1% in CHCl.sub.3).
EXAMPLE 14
Production of (R)-.alpha.-methyl-2-oxiranemethanol
816 mg (3.13 mmol) of (R)-2,3-dihydroxy-2-methylpropyl tosylate are
dissolved in 50 ml of diethyl ether and the solution is treated
portionwise with 1.08 g (9.68 mmol) of sodium tert.butylate. After
stirring for 3 hours 100 .mu.l (5.55 mmol) of water are added thereto and
the mixture is stirred for a further 30 minutes. The resulting potassium
tosylate is then filtered off and the filtrate is concentrated cautiously
(readily volatile epoxide). The residue is purified by chromatography over
silica gel 60 (0.040-0.063 mm, 20 g) using n-pentane/diethyl ether (in one
direction gradually varying ratio of the two components) as the eluent.
After careful concentration of the fraction there are obtained 72 mg of
crude (R)-.alpha.-methyl-2-oxiranemethanol. The determination of the
enantiomeric purity by capillary gas chromatography (chiral permethylated
.beta.-cyclodextrin phase) gives 99% ee of the (R)-isomer (reference
comparison).
This means that the alcohol produced under the influence of the enzyme (see
Examples 3-9 and 15) has the S-configuration, as in the case of the
unreacted ester.
EXAMPLE 15
Manufacture of {(S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methanol (asymmetric
hydrolysis of {(R,S)-2,2,4-trimethyl-1,3-dioxolan-4-yl}methyl butyrate
using an immobilized enzyme)
(i) Partial purification of Lipase P-30: The purification is effected
according to the method of M. Sugiura et al. [Biochim. Biophys. Acta 488,
353-358 (1977)]: 12.5 g of Lipase P-30 are taken up in 100 ml of sodium
acetate buffer (10 mmol, pH 7.0) and the mixture is stirred at 4.degree.
C. for half an hour. The insoluble material is removed by centrifugation
(43500 g, 20 min., 5.degree. C.) and the solution remaining is diluted to
100 ml with a further amount of buffer, whereby the enzyme activity
amounts to 2600 U/ml [for the measurement of this activity, 250 .mu.l of
tributyrin are emulsified in 25 ml of 0.1M sodium chloride solution and 1
ml of 0.1M sodium phosphate buffer (pH 7.0) and the reaction is initiated
by the addition of the enzyme sample; the course of the reaction is
monitored while maintaining a pH value of 7.0 by adding 0.1N sodium
hydroxide solution]. 20.9 g of ammonium sulfate (35% saturation) are added
to the solution within 30 minutes and the thus-formed suspension is
stirred at 0.degree. C. for a further 2.5 hours. Then the suspension is
centrifuged as described above and the sediment (about 1 percent of the
enzyme activity in the supernatant) is dissolved in a small amount of
sodium phosphate buffer (50 mmol, pH 7.5) and dialyzed within 16 hours
against 4.times.1 l of the same buffer at 4.degree. C. [Spektra/Por,
cut-off limit of the molecular weight 3500 (Spectrum Medical Industries,
Los Angeles, Calif., USA)]. This gives 14.7 ml of a concentrated solution
of Lipase P-30 (1.33.multidot.10.sup.4 U/ml; enzyme activity measured as
described above).
(ii) Immobilization of lipase P-30: 500 .mu.l of the lipase solution are
diluted four times with the phosphate buffer described above and 500 mg of
Eupergit C beadlets (Rohm, Weiterstadt, FRG) are added thereto. The
suspension is then shaken gently at room temperature for 63 hours and the
beadlets are subsequently filtered off, washed with 0.1M sodium chloride
solution (about 0.1 percent of the enzyme activity in the filtrate) and
dried at 16 mbar for 15 minutes. In this manner there are obtained 1.36 g
of moist beadlets (234 U/g, this enzyme activity is measured as described
above; stored at 4.degree. C. for a few days).
(iii) Asymmetric hydrolysis: 10,0 g (46.2 mmol) of {(RS) 2,2,4
trimethyl-1,3-dioxolan-4-yl}methyl butyrate are emulsified in 185 ml of
0.1M sodium chloride solution and 5 ml of 0.1M sodium phosphate buffer (pH
7.5). After adjusting the pH value to 7.5 with 1.0N sodium hydroxide
solution the reaction is initiated by the addition of 500 mg of moist
Eupergit beadlets. The pH value is held constant at 7.5 by stirring in
1.0N sodium hydroxide solution. After the addition of a total of 22.2 ml
(22.2 mmol; 48 percent reaction conversion after 6.9 hours) of titrating
agent the enzyme beadlets are filtered off and washed five times with 10
ml of 0.1M sodium chloride solution each time. The filtrate is combined
with the first washing solution and the combined aqueous phase is
extracted four times with 200 ml of methylene chloride each time. The
combined organic phases are then dried over anhydrous sodium sulfate and
concentrated at 600 mbar/40.degree. C. The residue is subjected to a
column chromatography using silica gel 60 (0.040-0.063 mm) and
n-hexane/ethyl acetate (2:1). In this manner there are obtained 3.02 g
(20.7 mmol, 44.7% of the theoretical yield) of the title compound which by
gas chromatography is found to be more than 95% pure and which has a
percentage ee of more than 99%.
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